The objective of the proposed studies is to investigate the effect of lipid on the proliferation and differentiation of normal rat mammary epithelial cells (rmec), with the long-term goal of understanding the mechanism by which diets high in polyunsaturated fat stimulate mammary tumorigenesis. It is proposed that alterations of membrane phospholipid composition modulate the receptor transducing mechanism for hormones and growth factors, thus affecting cell proliferation and differentiation. Specific emphasis will be placed on protein kinase C, since this enzyme, which plays a major role in intracellular signal transduction, requires lipid for its activity. These studies will be carried out using a primary culture model system recently developed in our laboratory in which rat mammary epithelial cells obtained from immature virgin rats are grown within a reconstituted basement membrane matrix in a serum-free defined medium. This model allows for both extensive proliferation of the mammary epithelial cells (15-30 fold within 14-21 days) as well as functional and morphological differentiation equivalent to that of the lactating mammary gland. AIM 1. To carry out a comprehensive study of the effects of specific hormones and growth factors on morphological and functional differentiation of normal rat mammary epithelial cells in primary culture, with the goal of completing characterization of this unique model system. These studies will serve as the foundation upon which the effects of lipid as well as other protein kinase C modulators can be investigated. AIM 2. To continue studies on the effects of lipid on proliferation as well as morphological and functional differentiation of normal rat mammary epithelial cells. These studies will focus on the hormonal and growth factor conditions under which fatty acids and other lipids modulate specific mammary epithelial cell functions, with the goal of identifying those growth factors whose activity can be regulated by changes in membrane lipid composition and/or by the lipid in the incubation medium, and the mechanism by which this action is mediated. AIM 3. To determine the role of specific protein kinase C isoenzymes in the proliferation and differentiation of normal rat mammary epithelial cells. In these studies, protein kinase C will be monitored during development of both morphological and functional differentiation, as well as in response to specific combinations of hormones and growth factors known to favor either the differentiation or proliferation of the mammary cells. Considerable emphasis will be placed on defining the effect of individual hormones and growth factors on the activity and isoenzyme composition of protein kinase C and the effect of protein kinase C inhibitors and activators on proliferation and differentiation. AIM 4. To determine if changes in membrane phospholipid composition affect receptor transducing mechanisms, with specific emphasis on protein kinase C. In this aim, the effect of membrane lipid on hormone- or growth factor-induced translocation of protein kinase C, as well as on its ability to phosphorylate specific protein(s) will be assessed.